Methods for monitoring transcription or translation and their uses in drug screening are known in the art. For example, U.S. Pat. No. 5,811,231 discloses methods for measuring transcription or translation levels from genes linked to native eukaryotic stress promoters, especially those of mammals. The methods yield information concerning the action of a compound on a subcellular level which may be utilized to design antitoxins.
U.S. Pat. No. 5,569,588 discloses methods and compositions for estimating the physiological specificity of a candidate drug. The methods use one reporter in the cells and compare reporter gene product signals from each cells before and after contacting with candidate drug to obtain a drug response profile.
U.S. Pat. No. 5,965,352 discloses methods for determining the primary and secondary biological pathways through which a drug acts on a cell, and identifying the proteins and genes which are affected via each pathway. The method involves comparing measurements of RNA or protein abundances or activities in response to drug exposure with measurements of RNA or protein abundances or activities in pathways possibly affected by the drug in response to controlled, known perturbations of each pathway. Further, the invention provides methods for comparing the effects of two different drugs by comparing measurements of RNA or protein abundances in response to exposure to a first drug with RNA or protein abundances in response to exposure to another drug or drugs.
U.S. Pat. No. 6,046,002 discloses methods for identifying gene products that mediate a phenotype, such as drug resistance or sensitivity, as well as methods for identifying new bioactive compounds, by detecting differences in growth (e.g., as measured by growth rate) between host cells that differ in target gene product dosage (e.g., two copies of a target gene product-encoding sequence compared to one copy). Specifically, the invention features a method for identifying a nucleotide sequence encoding a target gene product of a bioactive compound by: a) culturing a first host cell and a second host cell in the presence of a bioactive compound, wherein the first host cell contains a target gene product-encoding sequence expressed at a first expression level, and the second host cell contains the target gene product-encoding sequence expressed at a second expression level, wherein the second expression level is less than the first expression level; and b) comparing growth rates of the first host cell and the second host cell. An alteration (e.g., increase or decrease) in growth rate of the second host cell relative to the growth rate of the first host cell indicates that the expression level of the candidate target gene product-encoding sequence is a determinant of resistance or sensitivity to the bioactive compound and the candidate target gene product-encoding sequence encodes a target gene product of the bioactive compound.
However, the above-described methods are based on one-reporter and two measuring points within these ranges, namely no exposure or perturbation and fully saturating exposure or perturbation, or one-reporter and perturbation strengths and drug exposure levels.
U.S. Pat. No. 5,807,522 discloses a method of detecting differential expression of each of a plurality of genes in a first cell type, with respect to expression of the same genes in a second cell type. In practicing the method, there is first produced fluorescent-labeled cDNAs from mRNAs isolated from the two cells types, where the cDNAs from the first and second cell types are labeled with first and second different fluorescent reporters. A mixture of the labeled cDNAs from the two cell types is added to an array of polynucleotides representing a plurality of known genes derived from the two cell types, under conditions that result in hybridization of the cDNAs to complementary-sequence polynucleotides in the array. The array is then examined by fluorescence under fluorescence excitation conditions in which (i) polynucleotides in the array that are hybridized predominantly to cDNAs derived from one of the first or second cell types give a distinct first or second fluorescence emission color, respectively, and (ii) polynucleotides in the array that are hybridized to substantially equal numbers of cDNAs derived from the first and second cell types give a distinct combined fluorescence emission color, respectively. The relative expression of known genes in the two cell types can then be determined by the observed fluorescence emission color of each spot. Obviously, this is a laborious procedure and very difficult to test thousands bioactive compounds with thousands different cells. Also there are discrepancies between duplicate experiment results, caused by multiple manipulating steps and technical limitations of DNA microarray technology. Therefore, there is still a need for an assay that has time and cost-saving features.
It would be desirable to have a screening method that are capable of screening large number, e.g., thousands, of drug candidates simultaneously and capable of providing accurate and reliable information about the actual target(s) of the drug or candidate drug. It would also be desirable to have a screening method that is fast and cost-effective. The present invention addresses these and other related needs in the art.